The hair cell, located in the inner ear, is an essential component of the hearing process. The actin cytoskeleton plays a major role in hair cell function, through both the actin filament bundle in the stereocilium and the actin web in the cuticular plate. Six mutations have been found in gamma nonmuscle actin (DFNA 20/26) that cause autosomal dominant non-syndromic hearing loss. However, the molecular basis for the deafness caused by these mutations is unknown, in large part because of the inability to secure enough of the mutant actins to assess the effects of the mutations on actin function at the biochemical level. To address this problem and to understand the biochemical consequences of these mutations, we have cloned each mutation into yeast actin which is 91% identical to human gamma nonmuscle actin, and we can purify sufficient quantities of each of the mutant actins for biochemical analysis. These mutations cause allele-specific effects in yeast, but only one of the mutations affects polymerization of pure actin in vitro. This result suggest that the mutations may interfere, instead with actin's ability to be controlled by different actin filament regulatory proteins. We will use well-established biochemical and fluorescence microscopic assays of actin polymerization to assess the effects of these six deafness-causing mutations on actin's interaction with actin-binding proteins likely to to regulate actin filament function in the hair cell. Specifically, we will continue to use the yeast actin system to examine the mutations' effects on cofilin, an actin filament severing protein, Arp2/3 complex and formins which initiate actin polymerization and control actin filament length. We will examine the mutations' effects on actin's ability to interact with three deafness-associated actin filament bundling proteins, and espin, 1- catenin and Eps8. We will also use mass spectroscopy coupled with hydrogen-deuterium exchange to determine directly the effects of the mutations on actin filament conformation and flexibility. Finally, we will establish a baculovirus expression system to produce mammalian nonmuscle 3-isoactins containing the deafness mutations to allow assessment of the effects of the mutations in the actin in which they actually occur in the hair cell. RELEVANCE: Deafness is a major health problem in the United States. The proposed work will provide insight into the biochemical basis for the deafness caused by these six actin mutations. It will lead to a rigorous systematic examination of the properties of pure 3-nonmuscle isoactin. Additionally, the information gained on how these mutations affect actin's interaction with actin regulatory proteins will provide valuable new information concerning how actin functions and is regulated at the biochemical level in the normal ear. Thus, the work should enhance our understanding of the molecular basis of the normal hearing process. NARRATIVE: The proposed work investigates the effects on actin function of mutations in actin that lead to deafness. The results will provide insight into the molecular basis of the deafness these mutations cause as well as provide insight into the molecular mechanisms that govern normal hearing in humans.